Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

GIWAXS experimental methods at the NFPS-BL17B beamline at Shanghai Synchrotron Radiation Facility.

The BL17B beamline at the Shanghai Synchrotron Radiation Facility was first designed as a versatile high-throughput protein crystallography beamline and one of five beamlines affiliated to the National Facility for Protein Science in Shanghai. It was officially opened to users in July 2015. As a bending magnet beamline, BL17B has the advantages of high photon flux, brightness, energy resolution and continuous adjustable energy between 5 and 23 keV. The experimental station excels in crystal screening and structure determination, providing cost-effective routine experimental services to numerous users. Given the interdisciplinary and green energy research demands, BL17B beamline has undergone optimization, expanded its range of experimental methods and enhanced sample environments for a more user-friendly testing mode. These methods include single-crystal X-ray diffraction, powder crystal X-ray diffraction, wide-angle X-ray scattering, grazing-incidence wide-angle X-ray scattering (GIWAXS), and fully scattered atom pair distribution function analysis, covering structure detection from crystalline to amorphous states. This paper primarily presents the performance of the BL17B beamline and the application of the GIWAXS methodology at the beamline in the field of perovskite materials.

A high-affinity RBD-targeting nanobody improves fusion partner's potency against SARS-CoV-2.

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

Utilization of AlphaFold2 to Predict MFS Protein Conformations after Selective Mutation.

The major facilitator superfamily (MFS) is the largest secondary transporter family and is responsible for transporting a broad range of substrates across the biomembrane. These proteins are involved in a series of conformational changes during substrate transport. To decipher the transport mechanism, it is necessary to obtain structures of these different conformations. At present, great progress has been made in predicting protein structure based on coevolutionary information. In this study, AlphaFold2 was used to predict different conformational structures for 69 MFS transporters of E. coli after the selective mutation of residues at the interface between the N- and C-terminal domains. The predicted structures for these mutants had small RMSD values when compared to structures obtained using X-ray crystallography, which indicates that AlphaFold2 predicts the structure of MSF transporters with high accuracy. In addition, different conformations of other transporter family proteins have been successfully predicted based on mutation methods. This study provides a structural basis to study the transporting mechanism of the MFS transporters and a method to probe dynamic conformation changes of transporter family proteins when performing their function.

In Situ Formation of Hierarchical Bismuth Nanodots/Graphene Nanoarchitectures for Ultrahigh-Rate and Durable Potassium-Ion Storage.

Metallic bismuth (Bi) has been widely explored as remarkable anode material in alkali-ion batteries due to its high gravimetric/volumetric capacity. However, the huge volume expansion up to ≈406% from Bi to full potassiation phase K3 Bi, inducing the slow kinetics and poor cycling stability, hinders its implementation in potassium-ion batteries (PIBs). Here, facile strategy is developed to synthesize hierarchical bismuth nanodots/graphene (BiND/G) composites with ultrahigh-rate and durable potassium ion storage derived from an in situ spontaneous reduction of sodium bismuthate/graphene composites. The in situ formed ultrafine BiND (≈3 nm) confined in graphene layers can not only effectively accommodate the volume change during the alloying/dealloying process but can also provide high-speed channels for ionic transport to the highly active BiND. The BiND/G electrode provides a superior rate capability of 200 mA h g-1 at 10 A g-1 and an impressive reversible capacity of 213 mA h g-1 at 5 A g-1 after 500 cycles with almost no capacity decay. An operando synchrotron radiation-based X-ray diffraction reveals distinctively sharp multiphase transitions, suggesting its underlying operation mechanisms and superiority in potassium ion storage application.

Ultrasound-guided totally implantable venous access ports via the right innominate vein: a new approach for patients with breast cancer.

BACKGROUND: To evaluate the feasibility and safety of ultrasound-guided totally implantable venous access port (TIVAP) implantation via the right innominate vein in patients with breast cancer. METHODS: Sixty-seven breast cancer patients underwent ultrasound-guided implantation of TIVAPs via the right innominate vein for administration of chemotherapy. Clinical data including technical success, success rate for the first attempt, periprocedural, and postoperative complications were recorded and retrospectively studied. RESULTS: All patients underwent successful surgery. The success rate of the first attempt was 95.52% (64/67). The operation time was 28 to 45 min, with an average of 36 ± 6 min. Periprocedural complications included artery punctures in 1 (1.50%, 1/67) patient. Prior to this study, the mean TIVAP time was 257 ± 3 days (range 41 to 705 days). The rate of postoperative complications was 4.48% (3/67), including catheter-related infections in 1 case and fibrin sheath formation in 2 cases. Up to the present study, three people had unplanned port withdrawal due to complications, and the TIVAPs for 25 patients were still in normal use. CONCLUSIONS: The success rate of ultrasound-guided TIVAPs via the right innominate vein is high with low complications, thus safe and feasible. This technique can provide a new option for chemotherapy of breast cancer patients.

Structural basis of nonribosomal peptide macrocyclization in fungi.

Nonribosomal peptide synthetases (NRPSs) in fungi biosynthesize important pharmaceutical compounds, including penicillin, cyclosporine and echinocandin. To understand the fungal strategy of forging the macrocyclic peptide linkage, we determined the crystal structures of the terminal condensation-like (CT) domain and the holo thiolation (T)-CT complex of Penicillium aethiopicum TqaA. The first, to our knowledge, structural depiction of the terminal module in a fungal NRPS provides a molecular blueprint for generating new macrocyclic peptide natural products.

Upgrade of crystallography beamline BL19U1 at the Shanghai Synchrotron Radiation Facility.

BL19U1, an energy-tunable protein complex crystallography beamline at the Shanghai Synchrotron Radiation Facility, has emerged as one of the most productive MX beamlines since opening to the public in July 2015. As of October 2023, it has contributed to over 2000 protein structures deposited in the Protein Data Bank (PDB), resulting in the publication of more than 1000 scientific papers. In response to increasing interest in structure-based drug design utilizing X-ray crystallography for fragment library screening, enhancements have been implemented in both hardware and data collection systems on the beamline to optimize efficiency. Hardware upgrades include the transition from MD2 to MD2S for the diffractometer, alongside the installation of a humidity controller featuring a rapid nozzle exchanger. This allows users to opt for either low-temperature or room-temperature data collection modes. The control system has been upgraded from Blu-Ice to MXCuBE3, which supports website-mode data collection, providing enhanced compatibility and easy expansion with new features. An automated data processing pipeline has also been developed to offer users real-time feedback on data quality.

Structural and functional characterization of itaconyl-CoA hydratase and citramalyl-CoA lyase involved in itaconate metabolism of Pseudomonas aeruginosa.

Itaconate is a key anti-inflammatory/antibacterial metabolite in pathogen-macrophage interactions that induces adaptive changes in Pseudomonas aeruginosa-exposed airways. However, the impact and mechanisms underlying itaconate metabolism remain unclear. Our study reveals that itaconate significantly upregulates the expression of pyoverdine in P. aeruginosa and enhances its tolerance to tobramycin. Notably, the enzymes responsible for efficient itaconate metabolism, PaIch and PaCcl, play crucial roles in both utilizing itaconate and clearing its toxic metabolic intermediates. By using protein crystallography and molecular dynamics simulations analyses, we have elucidated the unique catalytic center and substrate-binding pocket of PaIch, which contribute to its highly efficient catalysis. Meanwhile, analysis of PaCcl has revealed how interactions between domains regulate the conformational changes of the active sites and binding pockets, influencing the catalytic process. Overall, our research uncovers the significance and mechanisms of PaIch and PaCcl in the efficient metabolism of itaconate by P. aeruginosa.

Alleviating allergic airway diseases by means of short-term administration of IL-2 and dexamethasone.

BACKGROUND: IL-2 combined with dexamethasone can upregulate regulatory T (Treg) cells, but the mechanism is still under exploration. OBJECTIVE: Although previous studies focused on upregulating Treg cells in normal mice, here we investigated whether the IL-2 and dexamethasone combination treatment can upregulate Treg cells in pathological conditions, specifically in alleviating allergic airway disease. We also examined the potential pathway involved in Treg cell upregulation by IL-2 and dexamethasone. METHODS: We evaluated the dose of IL-2 and dexamethasone required to upregulate Treg cells in vivo and in vitro. We also tested IL-2 and dexamethasone in the intervention of allergic airway disease in a murine model. RESULTS: We found that administration of 400,000 IU of IL-2 and 0.1 mg of dexamethasone per mouse was effective in upregulating Treg cells, as well as in alleviating allergic airway disease in an established animal model, but this phenomenon disappeared after anti-CD25 antibody administration. We discovered that an in vitro low dose of IL-2 can protect Treg cells did not protect CD4(+)CD25(-) cells from dexamethasone-induced apoptosis by affecting forkhead box O3a phosphorylation through the Akt and serum and glucocorticoid-induced protein kinase pathways. CONCLUSIONS: IL-2/dexamethasone treatment can alleviate existing allergic airway diseases by upregulating Treg cells in vivo. A low dose of IL-2 (10(-9) to 10(-11) mol/L) can protect Treg cells but not CD4(+)CD25(-) cells from dexamethasone-induced apoptosis in vitro, thereby explaining a possible mechanism of increased proportion of Treg cells.

Perioperative and Postoperative Complications of Supraclavicular, Ultrasound-Guided, Totally Implantable Venous Access Port via the Brachiocephalic Vein in Adult Patients: A Retrospective Multicentre Study.

PURPOSE: The totally implantable venous access port (TIVAP) provides patients with safe, effective and long-term convenient venous access for the administration of medications such as chemotherapy drugs. The implantation and long-term use of TIVAP are related to thrombosis, infection and other complications. In this study, the medical records of multicentre patients were collected, and the perioperative and postoperative complications were retrospectively analysed to objectively evaluate the safety of the implantation of supraclavicular, ultrasound-guided TIVAP via the brachiocephalic vein (BCV). PATIENTS AND METHODS: We retrospectively analysed the clinical data of 433 adult patients who had undergone ultrasound-guided TIVAP implantation via the BCV at four hospitals in China from March 2018 to May 2019. The success rates of the first puncture, operation time, and perioperative and postoperative complications were analysed. RESULTS: All the TIVAPs were implanted successfully (100%). The average TIVAP carrying time was 318.15 ±44.22 days (range: 38-502 days) for a total of 197,694 catheter days. The success rate of the first puncture was 94.92% (411/433), and the average operation time was 29.66 ±7.45 min (range: 18-60 min). The perioperative complications included arterial puncture in 4 patients and pneumothorax in 1 patient. The incidence of postoperative complications was 5.08% (22/433), including poor incision healing (n = 2), catheter-related infection (n = 3), port infection (n = 6), thrombosis (n = 2) and fibrin sheath formation (n = 8). Another patient had infusion disturbance 2 days after the operation, and chest X-ray showed bending at the connection between the catheter and port. No other serious complications occurred, such as catheter rupture and drug leakage. The total incidence of complications was 6.24% (27/433). CONCLUSION: This study showed excellent tolerance of supraclavicular, ultrasound-guided BCV puncture to implant TIVAP and a low incidence of complications. As a safe and effective method of TIVAP implantation, it can provide a new choice for clinicians.

Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody.

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1-6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7-17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 µg mL-1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

A Synthetic Human Antibody Antagonizes IL-18Rß Signaling Through an Allosteric Mechanism.

The interleukin-18 subfamily belongs to the interleukin-1 family and plays an important role in modulating innate and adaptive immune responses. Dysregulation of IL-18 has been implicated in or correlated with numerous diseases, including inflammatory diseases, autoimmune disorders, and cancer. Thus, blockade of IL-18 signaling may offer therapeutic benefits in many pathological settings. Here, we report the development of synthetic human antibodies that target human IL-18Rß and block IL-18-mediated IFN-γ secretion by inhibiting NF-κB and MAPK dependent pathways. The crystal structure of a potent antagonist antibody in complex with IL-18Rß revealed inhibition through an unexpected allosteric mechanism. Our findings offer a novel means for therapeutic intervention in the IL-18 pathway and may provide a new strategy for targeting cytokine receptors.

A glucocorticoid amplifies IL-2-induced selective expansion of CD4(+)CD25(+)FOXP3(+) regulatory T cells in vivo and suppresses graft-versus-host disease after allogeneic lymphocyte transplantation.

Regulatory T (Treg) cells are a subpopulation of T cells that not only prevent autoimmunity, but also control a wide range of T cell-dependent immune responses. Glucocorticoid treatment (dexamethasone, or Dex) has been reported to amplify IL-2-mediated selective in vivo expansion of Treg cells. We simultaneously administered Dex and IL-2 to the donor in a murine allogeneic lymphocyte transplantation model to expand functional suppressive CD4(+)CD25(+)FOXP3(+) T cells in the graft and to raise the regulatory T cell/effector T cell (Treg/ Teff ) ratio to prevent graft-versus-host disease (GVHD). After combined treatment of the donor with Dex (5 mg/kg/day) and IL-2 (300,000 IU/mouse/day) for 3 days, grafts were subjected to flow cytometric analysis, and transplantation was carried out from male C57BL/6 mice to female BALB/c mice aged 8-12 weeks. Results showed that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive CD4(+)CD25(+)FOXP3(+) T cells in the murine spleen. In this murine allogeneic transplantation model, the grafts from donors with Dex and IL-2 pre-treatment led to a longer survival time for the recipients than for the control group (median survival time > 60 day vs. 12 day, P=0.0002). The ratio of Treg/Teff also increased remarkably (0.43+/-0.15 vs. 0.14+/-0.01, P=0.01). This study demonstrated that co-stimulation with Dex and IL-2 selectively expanded functional CD4(+)CD25(+)FOXP3(+) T cells in vivo, and that grafts from donors pre-treated with Dex and IL-2 led to longer survival time and greater suppression of GVHD after allogeneic transplantation. Thus, GVHD can be suppressed by the specific expansion of regulatory T cells with Dex and IL-2 in graft donors.

BMPRII is a direct target of miR-21.

MicroRNAs (miRNAs) are a type of small non-coding RNAs that regulate cognate mRNA expressions at the post-transcriptional stage. Although several miRNAs are known to be involved in various biological processes, including developmental timing, patterning, embryogenesis, differentiation and organogenesis, growth control, and apoptosis, many target genes and the functions of most miRNAs are still unclear. Since there is only a partial complementarity between miRNAs and their targets in animal cells, it is difficult to identify the specific target genes for a given miRNA and elucidate its function. In this study, we confirmed that bone morphogenetic protein receptor II (BMPRII) is a direct target of miR-21, and also showed that the protein level of BMPRII correlates inversely with the amount of miR-21 in PC3 and Lncap cells. These findings suggest that miR-21 may have a potential role in regulating the malignancy and metastatic abilities of prostate cancer cells and in self-renewal of stem cells by regulating the expression of BMPRII.

MicroRNA expression profiling in diabetic GK rat model.

MicroRNAs (miRNAs), which are a newly identified class of small single-stranded non-coding RNAs, regulate their target genes via post-transcriptional pathway. It has been proved that miRNAs play important roles in many biological processes. To better understand miRNA function on type 2 diabetes, we used an oligonucleotide microarray to monitor miRNA expression profiles of Goto-Kakizaki (GK) and Wistar rats' skeletal muscle. It was found that seven miRNAs were downexpressed and two miRNAs were over-expressed in the muscle of GK rats. Among them, miR-24 showed the most prominent change. p38 MAPK, which is a direct target of miR-24, also showed expression difference. All the data give a clue that miR-24 might be associated with diabetes through down-regulation of p38 MAPK.

Anisotropic elasticity and plasticity of an organic crystal.

Organic crystals are generally considered to be brittle, inelastic materials, which pose challenges for application in flexible devices. Inspired by α helical proteins for their key structural role in flexible hair, here, we describe the construction of a spring-like hydrogen bonded network through the self-assembly of a-OH/e-OH cyclohexanol derivatives.

Glucocorticoid-induced apoptosis requires FOXO3A activity.

Dexamethasone (DEX) induces apoptosis in lymphocytes, while protecting some cancer cells from apoptosis, by a poorly understood mechanism. In this study, we examined the potential role of the forkhead transcription factor (FOXO3A) in DEX-induced apoptosis. Unphosphorylated FOXO3A, the active form of FOXO3A, can translocate into nucleus and induce apoptosis. In lymphocytes, FOXO3A is upregulated by DEX treatment, while phospho-FOXO3A was downregulated. In several different types of cancer cells, we found that sensitivity to DEX correlated negatively to expression of phospho-FOXO3A. We conclude that DEX might maintain FOXO3A in its unphosphorylated, active form. Knockdown of FOXO3A expression using a small interfering RNA (siRNA) significantly reduces apoptosis in lymphocytes. This study suggests that FOXO3A has a pivotal role in DEX-induced apoptosis. Increased phospho-FOXO3A levels in cancer cells may explain, in part, their resistance to apoptosis. Therefore, FOXO3A may be a potential target for cancer therapy.